Abstract
Purpose:
Purpose: IL-27 and IL-35 are anti-inflammatory, heterodimeric cytokines in the IL-12 family of cytokines, which displays subunit and receptor promiscuity. IL-27 and IL-35 share subunit Ebi3, which is hypothesized to be responsible for the anti-inflammatory effects of IL-27 and IL-35. Ciliary neurotrophic factor (CNTF) is a nerve growth factor that has been shown to prevent neural cell damage. Though its effects on the immune system are unknown, it has been shown that CNTF can interact with receptors of the IL-6 family, many of which are homologous or shared by the IL-12 family cytokines. Our goal in this study is to genetically engineer recombinant CNTF and a covalently linked Ebi3 and CNTF fusokine and investigate whether they would have immunoregulatory functions in mouse models of CNS autoimmune diseases such as experimental autoimmune uveitis (EAU) and experimental autoimmune encephalomyelitis (EAE).
Methods:
Method: The CNTF/Ebi3 fusokine consists of mouse Ebi3 and CNTF linked by a 22 amino acids linker-peptide between the two subunits. The cDNA construct encoding the single chain CNTF or CNTF/Ebi3 fusokine construct was cloned into pMIB/V5-HIS vector with expression under the direction of the Baculovirus immediate-early promoter, OpIE2, which allows for high-level, constitutive expression of the proteins in insect cells. The expression vector was transfected into High Five insect cells and stably transfected clones were subjected to several cycles of Blasticidin S HCl selection.
Results:
Results: The secreted recombinant CNTF or Ebi3/CNTF has been partially purified by HIS-TAG affinity chromatography and size-exclusion chromatography using an FPLC system. The fusokine has been characterized by western blot analysis using antibodies against Ebi3 and the V5 protein tag. Consistent with the predicted molecular size, the Ebi3/CNTF fusokine is a protein at 54kDa.
Conclusions:
Conclusion: We have successfully bio-engineered a 54 kDa Ebi3/CNTF fusokine and demonstrated its efficient secretion in insect cell cultures. Large-scale, ex-vivo production of the Ebi3/CNTF fusokine will undoubtedly allow in-depth analysis of the effects of this novel fusokine on T cell differentiation and its therapeutic potential in murine models of uveitis (EAU) and multiple sclerosis (EAE).